Systems and methods for imaging and processing tissue

ABSTRACT

In accordance with preferred embodiments of the present invention, a method for imaging tissue, for example, includes the steps of mounting the tissue on a computer controlled stage of a microscope, determining volumetric imaging parameters, directing at least two photons into a region of interest, scanning the region of interest across a portion of the tissue, imaging a plurality of layers of the tissue in a plurality of volumes of the tissue in the region of interest, sectioning the portion of the tissue, capturing the sectioned tissue, and imaging a second plurality of layers of the tissue in a second plurality of volumes of the tissue in the region of interest, and capturing each sectioned tissue, detecting a fluorescence image of the tissue due to said excitation light; and processing three-dimensional data that is collected to create a three-dimensional image of the region of interest. Further, captured tissue sections can be processed, re-imaged, and indexed to their original location in the three dimensional image.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation application of U.S. application Ser.No. 13/297,035 filed Nov. 15, 2011 which claims priority to U.S.Provisional Application No. 61/413,543, filed Nov. 15, 2010, the entirecontents of the above applications being incorporated herein byreference.

BACKGROUND OF THE INVENTION

This invention addresses the pressing need in biomedical science forimaging and tissue processing methods which can quickly image multiplecomponents in thick tissues and whole organs with high molecularspecificity. In order to develop methods which achieve this goal, thechoice of an appropriate imaging technology is critical. Importantcriteria include spatial resolution, sensitivity, depth penetration,molecular specificity, data throughput and compatibility with the manybiochemical methods such as immunohistochemistry or FISH analysis. Manymethods to image 3D thick tissues have been developed, but most of thesetechniques do not have subcellular resolution or the necessary molecularsensitivity. For instance, while high resolution MRI is capable ofimaging whole animals, its resolution is limited to 10-100 μm and is notcompatible with common fluorescent markers. In comparison, opticalimaging methods in general provide the highest resolution andspecificity. Optical coherence tomography has a few-micron levelresolution and a penetration depth of a few millimeters, however,optical coherence tomography does not provide reliable subcellular levelimaging today. Optical projection tomography is more compatible withmolecular imaging and can study fluorescent and non fluorescentlystained tissues up to approximately 15 mm. However it also does notpossess sufficient resolution to resolve details of individual cells andhas difficulty in imaging tissues containing opaque materials such asbone or cartilage. Sheet plane illumination techniques, such asselective plane illumination microscopy (SPIM) has demonstrated theability to provide detailed images of over a millimeter of relativelytransparent samples such as embryos but, due to the residual scatteringthat exists even in optically cleared samples, SPIM and similartechniques have limitations with opaque samples and with samples whichhave an extent over several millimeters or which possess weakfluorescent signals. Further, since it is not generally possible toimmunostain whole organs due to the limited diffusivity of antibodiesand even of small molecules into intact tissues, sheet plane techniquesare of limited use without first sectioning the tissue to allow thepenetration of the appropriate labels. This restricts their use inanswering many biological questions which require either IHC or FISHanalysis to reveal relevant biomarkers.

Among 3D tissue optical imaging techniques, two-photon microscopy (TPM)is particularly promising. TPM is a fluorescent optical microscopytechnique. It features sub-micron resolution, low photo-toxicity,excellent penetration depth, and 3D sectioning capability. The excellentdepth penetration of TPM in tissues is due to lower scattering andabsorption of the infrared excitation wavelength employed and the lackof the need for a detection pinhole allowing greater signal collectionefficiency than in confocal microscopy. In addition, like allfluorescence based techniques, it provides high molecular specificity inmapping gene and protein expression profiles, and has clearlydemonstrated its utility for visualizing gene activity in vivo with GFPover the past decade.

While, two-photon microscopy (TPM) can image in highly scattering media,it is still limited to approximately less than a millimeter penetrationinto opaque samples. To overcome the depth penetration limitation oftwo-photon microscopy in studying thicker specimens, preferredembodiments of the inventions use an automated microtome integrated intoa high speed TPM system. By alternating and overlapping opticalsectioning with mechanical sectioning, it is possible to rapidly imagesamples with arbitrary thickness. Once the upper portion of the sampleis imaged, the uppermost portion of the tissue sample can be removed bythe microtome. A critical problem with mechanically sectioned tissues,however, is the difficulty in comparative analysis due to stretching,compressing and/or rotation of tissue structures caused by mechanicalsectioning. Prior methods have used fiducial markers formed by drillingholes or otherwise altering the tissue to aid in alignment.

While direct intravital tissue labeling, transgenic animals, nativetissue autofluorescence, and SHG contrast provide powerful means tovisualize the complex 3D biochemical environment within a tissue, thereare still a large number of biochemical states and signatures which areonly possible to examine by other means, such as immunohistochemistry(IHC) staining. Unfortunately it is very difficult or impossible toreliably IHC stain whole mount tissues greater than approximately 100microns in depth. This is due to the large size of antibodies used inIHC staining and their subsequent slow diffusion and steric hindrancewithin the tissue.

SUMMARY OF THE INVENTION

The present invention relates to systems and methods of acquiring athree dimensional image of a tissue sample formed from a plurality ofimages tissue layers, processing sections of the imaged tissue samplefollowed by imaging of the processed sections to form a second threedimensional image of the sample and analysis of the first image andsecond image to characterize the tissue sample.

The present invention relates to systems and methods including movingthe optical system, the stage moving, or the vibratome moving forgenerating images of whole organs or thick tissues. The system caninclude a transfer system to move tissue sections for storage andregistration as well as for further processing and imaging.

Despite the strengths of traditional TPM, it cannot be directly appliedto organ level imaging due to three factors. First, the depthpenetration of TPM is still limited to a few hundred microns for mosttissue types, presenting a barrier to imaging tissue or organs whichhave a substantive axial extent. Second, the typical field of view ofstandard TPM has a linear dimension of only a few hundred microns, thusrequiring a scanning procedure over an extended period that isimpracticable for whole organ imaging. Third, standard TPM systems havean image acquisition speed that is also prohibitively slow for imagingmacroscopic 3D tissues. A typical TPM system would require approximately60 days at a minimum to image a 5 mm cube of tissue at a resolution of 1cubic μm and 10 μs pixel residence time, for example. Therefore thereexists a great benefit to save the sections after they have been removedfrom the whole mount tissue for further biochemical analysis. Furtherexamples of analysis, but not limited to, are, FISH, mass spectrometry,imaging mass spectrometry, PCR, micro dissection. Afterwards, thisadditional information can be used independently or combined with theoriginal 3D image which was obtained to extract further biologicalinformation from the sample.

Preferred embodiments of the invention can include two-photon 3D tissueimage cytometer (or alternatively referred to as a whole mount tissuescanner) using multi-photon excitation. To overcome the depthpenetration limitation of two-photon microscopy in studying thickerspecimens, preferred embodiments of the inventions use an automatedmicrotome integrated into a high speed TPM system. By alternating andoverlapping optical sectioning with mechanical sectioning, it ispossible to rapidly image samples with arbitrary thickness. Once theupper portion of the sample is imaged, the uppermost portion of thetissue sample can be removed by the microtome. The depth of mechanicalsectioning is chosen to be less than the imaged depth ensuring that theregion of the sample that is subjected to mechanical sectioning has beenalready imaged.

An advantage of this methodology over block face techniques is that anydistortions that are introduced by the sectioning procedure do notintroduce artifacts in the reconstructed 3D volume since the cut planeis always pre-imaged. Further, imaging of overlapping regions betweensuccessive sections ensures accurate digital registration betweensections on a pixel by pixel basis. To overcome the limitations in dataacquisition speed, two methods of high speed two-photon microscopy canbe utilized. To overcome the field of view limitations of the objective,the robotic stage raster scans the sample allowing a larger image to beconstructed from a series of overlapping 3D volumes. After the sectionis removed with the microtoming procedure, the entire sample istranslated towards the objective. Unlike with serial sectionreconstruction, where alignment of successive z-sections is oftenambiguous, the integrated precision robotic stage insures highreliability in registering neighboring volumes both in the axial andradial direction. The microscope system is entirely automated andrequires no user intervention once the sample has been mounted.

The present invention enables comparative analysis of image data usingimages obtained both before and after sectioning. Thus, for example, atransgenic mouse engineered to express GFP in a cell if protein X isbeing expressed can be section, processed and imaged using IHC stainingto determine if protein Y is also being expressed. Thus, a particularcell can be determined to have two different biomarker characteristicsbased on different processing and imaging of the tissue sample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1G illustrate image data generated by systems and methods of thepresent invention.

FIG. 1H illustrates the imaging and cutting depths of a sample beingimaged in accordance with the invention.

FIGS. 2A-2F illustrate a imaging and processing systems in accordancewith preferred embodiments of the invention.

FIG. 3 illustrates a process sequence for imaging and sectioning oftissue in accordance with the invention.

FIGS. 4A-4B illustrate perspective and taps views of a sectioning toolin accordance with the invention.

FIGS. 5A-5B illustrate tissue sectioning parameters in accordance withpreferred embodiments of the invention.

FIG. 6 illustrates a process sequence of methods used in imaging tissue.

FIG. 7 illustrates a process sequence processing of a plurality ofimages of a tissue sample.

FIG. 8 illustrates a process sequence for analyzing image data.

FIG. 9 illustrates a system for distributing sectioned samples forfurther processing.

FIG. 10 illustrates a system for separating tissue sections for furtherprocessing.

FIG. 11 illustrate a sectioning sequence of a heart.

FIG. 12A-12C illustrate section analysis of an animal heart.

FIG. 13 are images of a vascular casting process.

FIGS. 14A-14B are images of an animal brain cast and analysis.

FIG. 15 illustrate a process for imaging, sectioning and analysis of ananimal brain.

FIG. 16A-16D illustrate an animal brain atlas.

FIG. 17A-17D illustrate use of adjustable resolution in imageprocessing.

FIG. 18 illustrates imaging and alignment.

DETAILED DESCRIPTION OF THE INVENTION

FIG. 1A-1G illustrates image data that can be acquired and processed inaccordance with the invention. An entire mouse heart was imaged with submicron resolution and multispectral detection as shown in FIG. 1A. Thissystem visualizes details at the subcellular level throughout the entireheart, revealing features of the nuclei, vessel architecture, mesoscalearchitecture such as cleavage planes in the heart, and the macroscopicmorphology of the heart chambers. This entire 3D data can span almostfive orders of magnitude. FIG. 1B shows autofluorescence of heart tissuewith labeled nuclei and vasculature wherein the scale bar is 100 μm.FIG. 1C shows cleavage planes of laminar sheets of sectional myocardium.FIG. 1D shows morphology of the 3D microvasculature. FIG. 1E showsnuclei from the myocytes, fibroblasts and endothelial cells lining thevasculature. FIG. 1F shows the section outlined in FIG. 1B where thearrow indicates space between successive cleavage planes. FIG. 1G showsa whole organ 3D image of a heart that can be imaged after processing asset forth in preferred embodiments of the invention.

FIG. 1H demonstrates the relation of the imaging plane 14 with respectto the tissue surface 10, surface cut plane 12, and maximum imagingdepth 16 in each imaged region (17, 18) of tissue. Imaging below the cutdepth in the second region 11 enables more precise registration in theformation of 3D images of whole organs or deep tissue samples as theoverlapping region 17 extends below the cut depth 15. This enablesregistration of the first image region (17 and 18) with the secondregion 11 which also includes overlap region 17.

FIGS. 2A-2F show systems used for multiphoton multifocal microscope(MMM). This system can image at over 600 frames per second andsignificantly deeper into tissues than prior systems. The systemperforms multispectral detection methods that have better sensitivityand throughput than existing multispectral imaging systems.

A traditional confocal or two-photon system requires approximately 60days to image an entire mouse heart at sub-micron resolution. Howevergiven the high speed imaging employed using the present system, thepresent system images this in 3.5 days and preferably in approximately2-5 hours. As and additional example, this system routinely generatesmouse brain atlases with 100 micron spacing of coronal sections at 1.4μm pixel sampling in approximately 4 hours.

FIG. 2A shows an imaging and processing system 40 in accordance with theinvention. A processor 42 is connected to system components includinglight source 48, a first PMT channel 50, second PMT channel 52, thirdPMT channel 54, (x,y) scanner 56, vertical scanner 58 that translatesobjective lens 60 relative to the tissue sample 65 (such as a wholeorgan mouse brain), sample motorized stage 62, microtome 64 with cuttingtool 66, tissue section transport system 68 which moves each section oftissue to a storage system 70, a processing system 72 that furtherprocesses the section and a second imaging system 74 that can be used incombination with the first imaging system 45 to generate images ofprocessed tissue. The images of the tissue, both before and aftersectioning, can be stored in memory 46 and displayed in various formatsas described herein on display 44. Additional details regarding imagingsystems and methods of using these systems are described in U.S. Pat.Nos. 7,372,985 and 7,724,937 and in U.S. application Ser. No. 11/442,702filed on May 25, 2006, the entire contents of these patents andapplication being incorporated herein by reference. Further details ofsystems and methods of detecting time resolved data used in conjunctionwith preferred embodiments of the invention are described inInternational Application No. PCT/US2009/060731, the entire contents ofwhich is incorporated herein by reference. The processor 42 can beprogrammed with software that operates the system components and thatprocesses image data as further described herein. The transport system68 can move tissue samples for further processing and imaging asdescribed herein. The processor 42 can then analyze image data frombefore and after sectioning to correlate and quantify data tocharacterize the tissue in detail. As sectioning can alter the surfacemorphology of the tissue, this can create difficulty in the process ofcorrelating details in tissue structure after further processing andimaging. Preferred embodiments provide methods for analyzing image datafrom before and after sectioning in combination to characterize tissue.

A multiphoton imaging system 100 can be used in preferred systems andmethods of the invention for imaging tissue before and after sectioning.Referring to FIG. 2B a further embodiment of the invention provides formulti color detection MMM 100 in the xz-plane. An array of 2×8 beams isgenerated by the micro lens array 140. The setup here is illustratedwith two 1×8 beam lines. The distance between the foci in each line isdetermined by the combination of the source beam configuration and themicro lens array 140. Two light beams are conducted through the microlens array 140 and intermediate optics L1 onto the focal plane of themicroscope in which they create two lines of 1×8 foci. For simplifiedvisualization, in FIG. 2D only 3 of the 16 beam traces in a 2×8 setupare illustrated. The full field is then scanned by the mirroroscillation of the scanning mirror 130, in which the scanning amplitudesneed to be adapted to the distances of the foci. On the detection side aholographic diffraction grating 192 is incorporated that diffracts themultiple wavelengths emitted from the sample onto the photo-multiplierarrays of two stacked multi-anode PMTs 120, 124 (FIGS. 2E, 2F). In thesetup the two multi-anode PMTs are stacked on top of each other, eachserving as a spectral detection device for one line of 1×8 foci. Thegrating 192 properties (pitch/inch) and the focal length of the focusinglens (L4), which determines the distance between the grating and themulti-anode PMT, have to be chosen in accordance to the anticipatedfluorescent probes used for staining the tissue sample. For thisembodiment, a transmission grating 192 is used. Nevertheless, comparableand/or better efficiency can be achieved in embodiments that use areflection grating or a prism. FIG. 2C illustrates the illumination fociand their scanning in the focal xy-plane. Scanning is indicated for twoarrays of 8 foci each. FIG. 2D shows the detection path of two beamsprojected in the yz-plane through grating 192 and lens L4 onto the stackof two AM-PMTs 120, 124. FIG. 2E shows the detection path projected inthe xz-plane, where the beams are depicted passing through grating 192and lens L4 with each of eight color bands being collected by the twoAM-PMTs 120 and 124. FIG. 2F illustrates the anodes of the multi-anodePMTs 120, 124 in the x/y plane, showing that the 8×8 anode arrays of thedetectors each detects one of the two 1×8 beam lines, where each 1×8beam line has been diffracted by the grating 192E into eight colorbands.

In order to cut the tissue, the system integrates a vibrating blademicrotome into the high speed whole mount tissue scanner to allowautomated retrieval of tissue sections for later processing such asimmunohistochemistry (IHC) staining of the sectioned tissue slices.Vibrating blade microtomes, a variation of the basic microtome, arewidely recognized as superior for cutting thick sections from soft orfresh tissue samples.

A vibrating blade microtome as the cutting device has severalattractions. First it can section very soft materials such as unfixedbrain tissue. Further, and in particular importance with regards to thisinvention, a vibrating blade microtome allows the preservation of tissuesections for later analysis, including IHC/FISH staining, massspectrometry, microarray analysis, re-staining of the tissue with otherdyes, and imaging with other modalities.

Further advantages of a vibrating blade microtome with regards toembedding in paraffin or other resins: 1. A vibrating microtome allowsthe tissue sample to embedded in an agarose gel, which has betterpreservation of GFP fluorescence as opposed to paraffin embedding: 2.Reduced tissue autofluorescence in comparison to paraffin embeddedtissues: 3. Paraffin embedding has been shown to be of lower quality intissue samples greater than 4-5 mm in extent, which can affect tissuemorphology: 4. It is difficult to obtain quality sections with vascularcasts that have been embedded in paraffin using a traditional microtomeor milling machine.

Preferred embodiments use a standard cryostat or microtome to sectionfrozen or paraffin embedded tissue, respectively, as the sectioningdevice. For instance, in cryosectioning, the key instrument is thecryostat, which is essentially a microtome inside a freezer. Themicrotome is capable of slicing sections as thin as 1 μm. The usualhistology slice is cut at 5 to 10 μm. The tissue specimen is placed on ametal chuck and frozen rapidly to about −20 to −30° C. The specimen isembedded in a gel like media known as optimal cutting temperaturecompound (OCT). At this temperature, most tissues become rock-hard.Usually a lower temperature is required for fat or lipid rich tissue,and an even lower temperature for skin. Each tissue has a preferredtemperature for processing but generally a temperature below 0° C. isrequired. Subsequently it is cut frozen with the microtome portion ofthe cryostat, the section is picked up on a glass slide and stained(usually with hematoxylin and eosin, the H&E stain). The preparation ofthe sample is much more rapid than with traditional histology techniques(around 10 minutes vs. 16 hours). However, the technical quality of thesections is much lower.

Further advantages of a vibrating blade microtome with regards toparaffin embedding: 1. It is difficult to obtain quality sections withPU4ii vascular casts that have been embedded in paraffin using atraditional microtome or milling machine: 2. Better preservation of GFPfluorescence for future studies employing transgenic animals: 3.Paraffin embedding of tissue has been shown to compromise GFPfluorescence: 4. Reduced tissue autofluorescence in comparison toparaffin embedded tissues: 5. Paraffin embedding has been shown to be oflower quality in tissue samples greater than 4-5 mm in extent, which canaffect tissue morphology.

In one embodiment of the vibrating blade microtome 200 (often referredto as a vibratome) a flexure stage 206 can be employed (FIGS. 4A-4B).Flexure stages are high performance mechanical bearings which allowmotion by the bending of a load element. Since they are constructed froma monolithic piece of metal, they have no internal moving parts and thushave excellent wear characteristics, and precise and repeatable rangesof motion. In general, motion control requires two separate functionalunits: a force generating unit, such as motor 204, to actuate the motionand a bearing unit, such as flexure arm 210 on which a blade holderassembly 208 is mounted, the arm 210 being attached to platform 212.This operates to constrain the motion to the desired trajectory. Flexurebearings use elastic deformation of materials to generate degrees offreedom to allow certain motions while minimizing all others. Theallowed motions are determined by the geometry cut into the material ofthe bearing. Furthermore, it is possible to highly constrain the rangeof motion along a chosen axis. In the application at hand, motion in thez direction (into or out of the tissue sample) can result in poorquality sectioning. The vibrating blade 202 will have a peak to peakamplitude on the order of 2 mm. It is desirable to have a surfaceflatness of 1 micron which corresponds to stringent 0.05% requirement inthe parasitic motion perpendicular to the cut plane. A flexure stageminimizes any such motions.

Vibrating blade microtomes introduce smaller cutting forces on thetissue because the back and forth motion of the blade presents aneffectively sharper cutting profile, while still maintaining thestructural strength and integrity of the blade in comparison to a bladewith an actual narrower profile. The five important parameters 400 forcutting tissue are shown in FIGS. 5A and 5B. V_(T) is the feed rate 406of the material through the blade, d is the thickness 408 of thesection, a is the tool cutting angle 404; F is the vibration frequencyof the blade motion, and A is the amplitude of the blade motion. Theamount of material removed per section is the following product:M _(Removed) =d*Area*V _(T)where Area is the cross sectional area of the tissue. The frequency andamplitude of the blade must be adjusted depending on the thickness ofthe tissue section, the feed rate, and the tool angle, and of thematerial properties of the tissue itself. In practice, good qualitysections over a wide range (30-150 μm) of section thickness can beachieved by adjusting the cutting angle, feed rate and vibrationfrequency.

The blade holder assembly shown in FIGS. 4A and 4B is connected to themounting platform through a flexure arm which allows motion along thedirection indicated by the red dotted arrow. The blade holder assemblymotion is actuated via a flexure linkage connected to the DC motor. Theflexure linkage transforms the rotary motion of the DC motor (MCDC3006S,MicroMo, Inc.) to a harmonic linear motion with a frequency equal to thefrequency of the motor. The flexure linkage is designed to minimize anyforces transmitted to the blade holder assembly which are not co-linearwith the desired linear motion of the blade. The frequency of thecutting motion can be set by adjusting the speed of the motor. Theamplitude of the motion can be specified by adjusting the connection ofthe flexure linkage to the DC motor through a slotted linkage on themotor. The razor blade (stock 2.5″×0.3″ carbon steel) is fastened to theblade holder assembly through a micro flexure which securely clamps therazor blade. The blade angle α can be adjusted by rotating the bladeholder and locking it to the blade holder assembly.

The microtome specifications are characteristic of the mechanicalresponse of the system and can be verified using a 2-axis laser rangefinder to monitor the motion of the blade and XYZ axis stage. TheMCDC3006S motor speed performance is operating using an integratedrotary encoder. The defect rate can be established by sequential cuttingof 254 sections of 100 micron thickness (1″ total depth). Five separateruns can be performed for a total of 1270 sections. The section surfacequality of a 5 mm×5 mm area can be verified by post-sectioning with 3Dtwo photon imaging of the surface of the heart tissue which will be enface stained with fluorescein. The resulting 2D surface image can fitand the RMS deviation of the average surface position calculated.

TABLE Milestones Tissue: Rabbit myocardial Microtome Specifications Feedrate 0.1-10 mm/s Vibration Frequency 25-200 Hz Vibration Amplitude 0.1-2mm Section Thickness 10-100 μm Blade angle 5-20 degrees Parasiticz-motion 1 μm Cutting Characteristics Surface Quality 2 μm RMS DefectRate <1 defect in 1270

The system and methods in U.S. Pat. No. 7,372,985 describes a techniquefor obtaining images of a thick tissue the entire contents of thispatent being incorporated herein by reference. Briefly, U.S. Pat. No.7,372,985 describes systems and methods that combine optical andmechanical sectioning to obtain images of whole mount thick tissues.

In this present invention, systems and methods are used to capture thetissue sections which are removed from the whole mount tissue, and tomap additional biochemical and morphological information onto the wholemount tissue by analysis of the captured tissue sections.

While direct intravital tissue labeling, transgenic animals, nativetissue autofluorescence, and SHG contrast provide powerful methods tovisualize the complex 3D biochemical environment within a tissue, thereare still a large number of biochemical states and signatures which areonly possible to examine by other methods, such as immunohistochemistry(IHC) staining. Unfortunately it is very difficult or impossible toreliably IHC stain whole mount tissues greater than approximately 100microns in depth. This is due to the large size of antibodies used inIHC staining and their subsequent slow diffusion and steric hindrancewithin the tissue. Therefore there exists a great benefit to save thesections after they have been removed from the whole mount tissue forfurther biochemical analysis. Further examples of analysis are, withoutlimitation, FISH, mass spectrometry, imaging mass spectrometry, PCR,micro dissection.

In addition, once the tissue has been sectioned, the tissue slice canoften be much more easily stained with additional dyes. For instance,even whole mount staining of, for instance, a mouse brain or heart, witha dye is generally not possible. In these cases, the exterior portion ofthe organ is often much more strongly stained while the inner portionsweakly or not stained at all due to the problem of diffusion of the dyethrough a thick tissue. This is even true for small molecule stains suchas DAPI or Hoescht 33442. Other important dyes include dyes used instandard H&E. By staining or re-staining the tissue after it has beensliced, it is possible to use both a far wider range of dyes and morehomogeneous staining, and generate more types of contrast to identifyvarious tissue constituents.

In addition, due to limitations or configuration of the imaging deviceused in the whole mount imaging procedure, it may be desirable to imagethe captured tissue slices on a separate type of imaging instrument. Forinstance, some dyes or intrinsic molecules are better imaged usingone-photon confocal microscopy rather than multiphoton, or vice versa.Other modality examples include OCT, CARS, SHG, STED wide field and timeresolved fluorescence.

One strategy is diagnostic comprehensive imaging of whole mount tissues,followed by comprehensive processing, imaging, and indexing everycaptured tissue section. Alternatively, the imaging process can befollowed by selective IHC/FISH staining of individual sections ofinterest. Another method is selective imaging of a region of interest(ROI) of a whole mount tissue followed by processing, imaging, andindexing of captured tissue sections.

FIG. 6 is a flowchart describing the steps 550 involved in capturing atissue section from a whole mount tissue. The sample is mounted 502,imaging parameters are selected 504. After the uppermost portion of atissue block has been imaged 506, 508, the uppermost portion is removed510 from the tissue with a sectioning device, such as a vibratome. Thissection is captured 512 by a tissue retrieval device and transferred toa suitable chamber for storage. The imaging and sectioning procedurecontinues such that at the end, not only are images of the tissueobtained, but also the sectioned tissues have been saved, indexed totheir original position in the tissue, and transferred to a collectionchamber for each tissue block. The system then performs furtherprocessing 514 as described herein.

FIG. 7 is a flowchart describing the process of processing the capturedtissue sections, re-imaging the tissue sections by a method or varietyof imaging methods, and then indexing these obtained images back to theoriginal images obtained from the imaging of the whole mount tissue. Inthis way, it becomes possible to recover additional information aboutthe biochemical state or morphology of the original tissue block whichwas not possible previously. Each section is selected 602, processed604, imaged 606, indexed 608 and analyzed in combination 610, such as byoverlaying.

In comparison to current state of the art practices, tissue sections maybe retrieved from a standard vibratome, but there exists no easy methodto relate the tissue sections back to the original, un-sectioned tissueblock. Serial section analysis, for instance, is currently in use bymany laboratories, but it has proven very difficult to generate, say, 3Ddatasets of IHC stained tissues due to the difficulty of processing andimaging post sectioned tissues. Problems include mechanical distortionof the tissue, and difficulty in aligning successive sections of atissue. The described invention allows, for instance, IHC stainedtissues to be morphed back to an original pristine tissue block whichserves as a reference. The process of registration can include rigidrotation and translation of each pixel in an image, morphing and/ornon-rigid transformation. A preferred method can include a scaleinvariant feature transform (SIFT) registration process. In thisprocess, a plurality of features contained in the overlap region (FIG.1H) are used to define a transformation to map pixels in apost-sectioning image to achieve stacking and alignment.

FIG. 8 is flowchart describing steps in the process 700 of taking theimaged tissue slices and registering them back to the original wholemount dataset. The image data is retrieved from memory 702, loadedsequentially into computer memory 704, processed an/or enhanced 706,registered to original unsectioned image data 708, optionally compressed710, stored 712, identify and/or quantify features of interest 714,summarize results (graphically) 716 and display 718 the data and resultson display 44 or transmit to remote locations via a network.

A preferred embodiment uses a tissue recovery unit 800 shows in FIG. 9for tissue slices 808 which have been removed from the tissue 802 by themicrotome system 806. A tube 812 can be used with a fluid flow 810 ormounted on a tape to move the sections from imaging container 804 tocollection container 814.

A major technical risk is the initial collection of the tissue slice andtransfer to a collection chamber. The unit captures and transfersindividual slices. In other embodiments, a rotating collection chambercan be incorporated which enables every slice to be saved in its ownindexed container.

During the tissue recovery process the tissue is sectioned from thewhole mount tissue block at a pre-specified thickness. During thesectioning process, the tissue sample is moved by the sample stage onwhich the bath is mounted toward the blade and the vibratome bladevibrates perpendicular to the line of motion. The transfer tube ismounted near the vibratome which ensures its fixed position in regardsto the tissue surface and blade during the cutting process. A flow ofliquid, such as saline solution, is flowed thru the tube to induce thesectioned tissue to travel along the transfer tube to a collectionchamber. After the sectioning is complete, the section is transferredalong the tube. and to the collection container. The flow liquid throughthe transfer tube is generated by a pump as shown in FIG. 9.

An example of capture slices from a tissue block are show in FIG. 10where coronal sections from a mouse brain have been transferred to awell plate array 820 with one section per well. The sections are readyto be further analyzed by a variety of techniques including IHC.

Angiogenesis is the sprouting of new blood vessels from existingvasculature. It is a major component of the two biggest killers in theUnited States today, heart disease and cancer. Despite the importance ofangiogenesis to biomedicine, angiogenesis therapies have been difficultto develop due to the lack of effective assays. Individual angiogenesisassays on the market focus on narrow aspects of angiogenesis and are toooften performed under in vitro conditions or in other model systems withlow physiological relevance. They fail to capture the complex, multistep3D nature of angiogenesis which is strongly dependent on interactingcomponents specific to the microscopic tissue under study. Additionallycurrent assays suffer from low throughput and are unsuitable forintegration into drug development programs.

This invention describes a novel angiogenesis assay, which is based on adevice with includes a vibratome, a tissue section capture device, andtissue labeling and analysis. Furthermore, the captured section can bereimaged and overlaid onto the original 3D tissue structure underinspection. This enables analysis of IHC stained thick tissue having adepth of several millimeters. Its advantages include high speed,automation, and physiological relevance. The invention can incorporatehigh speed multiphoton or confocal microscopy. It provides ex vivoimaging of macroscopic portions of tissue up to entire organs in smallmammalian models. It has both subcellular detail and multispectralresolution. The system can be used for pharmaceutical development whereit is necessary to screen tens to hundreds of late stage drug candidatesfor target validation and efficacy.

Cardiac ischemia the leading cause of death in the United States. Whileangiogenic therapies hold much promise for the treatment of ischemicheart disease, they have not delivered on this promise, mostly due tothe complex nature of the angiogenic response, particularly at themicroscopic level. We use this invention or assay by studying theangiogenic response of myocardial tissue to exogenously applied bFGF ininfarct models. Additionally, the system correlates the in situangiogenic response to the tissue collagen content, nuclei distribution,and hypoxia state.

Angiogenesis is the formation of new blood vessels from pre-existingvasculature. It is a central component of many diseases includingmyocardial ischemia, cancer, diabetic retinopathy, macular degeneration,and rheumatoid arthritis. Angiogenesis has been characterized as apotential ‘organizing principle’ in biology, where an understanding ofangiogenesis mechanisms will have broad therapeutic applicability acrossa range of diseases. It has been emphasized that the enormous potentialof angiogenesis based therapies stating, Angiogenesis research willprobably change the face of medicine in the next decades, with more than500 million people worldwide predicted to benefit from pro- oranti-angiogenesis treatments.

Unfortunately, the development of new angiogenesis therapies is plaguedby the ambiguity, low throughput, inconsistency, and importantly, thelow physiological relevance of current angiogenesis assays. As such,there exists an enormous biomedical need and market opportunity forbetter, quantitative assays which can provide insights that are readilytransferable to the clinical setting or the next round of the drugdevelopment cycle. Innovative highly relevant, tissue based assays canreplace many of the contemporary histopathological practices rooted intechniques decades old.

Ischemic heart disease is the leading cause of death in the UnitedStates. It is caused by reduced blood flow to myocardial tissue, leadingto reduced heart function. Angiogenic treatment by recombinant proteingrowth factors to induce revascularization has long been recognized asan attractive alternative to traditional bypass surgery. Angiogenicagents such as the fibroblast growth factor (bFGF) or the vascularendothelial growth factor (VEGF) families induce a strong angiogenicresponse in myocardial tissue in animal models Based on theseobservations, high hopes were placed on subsequent human clinicaltrials. Unfortunately results from these trials have proved ambiguousand mostly disappointing. Intracoronary and intravenous VEGF infusionslead to no difference with placebo at 60 days and only ambiguous resultsat 120 days. Similarly, intracoronary bFGF did not improve exercisetolerance or symptoms compared to placebo. Continuous deliverystrategies have only been slightly more encouraging. Intramyocardialinjections of bFGF alone or in heparin-alginate microspheres in patientsundergoing bypass grafting appeared to improve symptoms and capillarydensity, but these trials were small and confounders, such as the impactof the revascularization procedure itself, complicated results. It isbecoming evident that effective angiogenesis requires sustained presenceof an appropriate growth factor mixture in the local milieu or nascentcapillaries will tend to otherwise regress. Nevertheless, it is unclearwhether myocardial growth factor concentrations adequate forangiogenesis have in fact been sustained in each of the clinical trials.

Indeed, knowledge of the local pharmacokinetic processes governinguptake and distribution of growth factors in a highly vascularizedtissue such as myocardium is limited. The effects of mass transfer intoand convection within capillaries may so locally influence growth factorconcentrations that the rate of angiogenesis might bear no relation tomean myocardial drug concentrations.

Myocardial tissue itself changes in response to angiogenesis, and theconsequences of the continuous formation of blood vessels on the abilityof locally delivered drug to sustain nascent vessels have not beenexamined. As such, even the basic question of whether growth factors arebest administered in ischemic vs. healthy vs. borderline regions—orwhether site of administration is irrelevant—remains unanswered. Withthe surge in interest in therapeutic angiogenesis, characterizing thevascularization response across the myocardial wall becomes of pressingimportance.

The growth of the microvasculature in angiogenesis occurs in a complexseries of tightly coordinated events: 1) activation of endothelial cellswithin the existing vascular 2) disbanding of the capillary basal laminalining the vascular wall 3) capillary sprout formation and endothelialcell migration 4) extension of sprouts and vessel tubular formation byendothelial cell proliferation 5) vessel maturation including basementmembrane formation and recruitment of pericytes along the newly formedvascular and 6) initiation of blood flow and remodeling of theneovascular network. It is very difficult to study all these steps in asingle assay, and thus many different in vitro and in vivo angiogenesisassays have been developed, each hoping to capture an aspect of theangiogenic cascade.

Current angiogenesis assays on the market by examining the most popularin vitro and in vivo assays, and then review the current state of theart of histological analysis of angiogenesis.

In Vitro Assays:

Endothelial cells play a central role in angiogenesis and many in vitroassays are based on endothelial proliferation, migration or tubularformation in response to an angiogenic factor. The advantages of theseassays are their low cost, simple use, high reproducibility and easyquantification. They have proven valuable as early screening tools forangiogenic activity. However, endothelial 2D cell culture assays havesignificant drawbacks: First, endothelial cells are highlyheterogeneous. Phenotypic differences have been noted betweenendothelial cells taken from capillaries and those taken from largevessels. Inter-organ differences exist as well with endothelial cellsthat are part of the blood brain barrier as one example. Furthermore,flow and matrix conditions in culture can significantly affect resultsas well, and differences in endothelial cell karyotype and activationstate in culture have also been observed. Most importantly however,fundamentally in vitro assays are gross simplifications that fail tocapture the interacting nature of the endothelial cells with thesurrounding tissue stroma and thus must always be used in conjunctionwith in vivo assays if to be useful.

In Vivo Assays:

In vivo assays attempt to capture the complex spatial and biochemicalcharacter of the angiogenic response. The three most popular include theCAM, matrigel, and the corneal eye pocket assays. In the CAM assay,angiogenesis is monitored on the chick embryo CAM membrane. The assay issimple and inexpensive to conduct, and lends itself to large scalescreening. However it is sensitive to environmental conditions,particularly O₂ concentration, and it can be difficult to quantifyvascular growth due to the difficulty in visualizing new vesselsreliably. Additionally, inflammatory responses can confound results. 2)In the matrigel assay, liquid angiogenic agents such as a drug or tumorcells are injected into a liquid matrigel which is then subcutaneouslyinjected into an animal where it solidifies. Host cells and vascularpermeate into the gel and tare later removed and quantified by measuringhemoglobin content or by histological examination. The assay istechnically easy and quantitative. However disadvantages beyond expensethe ill-defined biochemical matrigel composition, inflammation andchamber geometry all make interpretation difficult and limit theusefulness of the assay 3) The cornea assay is based on the placement ofan angiogenic inducer into a corneal pocket in order to evoke vascularoutgrowth from the peripherally located limbal vasculature. Incomparison to other in vivo assays, this assay has the advantage ofmeasuring only new blood vessels, because the cornea is initiallyavascular, and the non-invasive monitoring of the angiogenic response.Significant drawbacks include inflammation at the injection site, thetechnical difficult nature of the surgery, and the atypical nature ofthe normally avascular cornea, all which affects the relevance of theassay.

Histological Examination:

Given the limitations in vitro and in vivo assays, many researchers haveopted for direct histological evaluation of angiogenesis. Tissues are 2Dsectioned and then IHC stained for endothelial cells along vessel whichserve as a marker for vasculature. In principle this can provide adirect route for quantifying the vasculature; however in practice itsuffers from significant limitations: 1. The IHC staining for theendothelial cells is not always effective or homogeneous, leading tosignificant ambiguity 2. Patent vessels cannot be discriminated from nonpatent vessels 2. Only 2D information is provided and vessels whichcross the examination plane multiple times cannot be taken into account3. Information about the 3D vascular network architecture is completelylost 4. The manual image inspection process is laborious 5. By thenecessity of the labor intensive nature, sparse sampling must beemployed. 6. Many months of training are required 7. Quantitativecomparison between different observers is difficult.

In summary, while the specific limitations of the current assays aremyriad, the underlying cause of these weaknesses is the complex natureof angiogenesis: a multistep 3D process occurring in situ on themicroscopic scale, with a spatially varying biochemicalmicroenvironment, and strong dependence on the interacting componentsspecific to the tissue under study. Further complicating matters, mostassays are based on simple measures of changes in vascular density orextent, despite therapeutic efficacy depending on microvascular patencyand interaction with the surrounding 3D tissue stroma as well. As aresult, current angiogenesis assays have serious shortcomings and areeither poor predictors of how well an angiogenic therapy will perform inthe clinical setting, or are too costly and lack necessary throughput.

Pharmaceutical companies are currently investing vast resources indeveloping new angiogenic drugs and therapies. It is currently estimatedthat the cost of developing a new drug is $802 million and takesapproximately 12 years (21). Given that the life of a patent is only 20years, pharmaceutical companies often have as a little as six to eightyears to recoup not only the costs of developing the drug but of alldrug candidates which did not make it through clinical trials. It hasbeen estimated that it costs a pharmaceutical company approximately $1million per day for every day that a successful drug is delayed tomarket. There is intense pressure to find new ways to reduce the costsand times to develop new drugs. In 2006 over 43 different drugs withanti-angiogenic effect were estimated to be in various stages ofdevelopment. Pro-angiogenic therapies are also actively being developedfor treating hypoxic tissues resulting from occluding lesions incoronary or cerebral arteries in the heart and brain respectively.

The assay described herein can be used in the late drug developmentcycle with animal model testing. It has the potential to providecrucial, contextual information at the physiologically relevant level tosee which drugs work, and just as importantly which drugs should beabandoned before the start of expensive clinical trials. Key stumblingblocks which have been holding back more tissue based studies are thelack of automation, high throughput and ease of use and mostimportantly, relevance.

Robust vascular segmentation requires large S/N (signal to noise ratio)levels. In high speed imaging, this can be challenging to achieve due tolow pixel residence times. Vasculature casting is an attractivealternative labeling protocols which label the vasculature wall. Itprovides far higher signal levels.

Additionally vibrating blade microtomes can section vascular casts.Vascular casting is an attractive method to image the morphology of avascular tree since polymer vascular casts have very high signal levelsin comparison to vascular wall labeling strategies. These high signallevels allow fast imaging with pixel residence times on the order of 0.5μs, but still obtain sufficiently high vascular signal levels forautomatic segmentation of the vascular network.

In vascular casting, a polymer is injected into the animal and fills theentire vascular tree and polymerizes into a solid. Since the entirevolume of the vessel is labeled and not just the vessel wall signallevels can be very high and thus suitable for automated segmentation.

Mercox and Batson's no. 17 are two of the most popular casting agentsbut lack inherent fluorescent contrast. Additive fluorescent dyes existfor the agents, but degrade in cast performance, preventing it frompenetrating into the smallest vessels. Additionally, they become verybrittle after curing. Initial tests have shown that mechanical stressimposed by whole mount milling or sectioning of tissues causes fracturesdeep within the tissue. The resulting discontinuities in the vasculaturetree make them an unfavorable choice for a casting agent. However, a newcasting material, PU4ii (vasQtec, Zurich) has been recently developed.It is well suited for fluorescence microscopy and has been shown to beeffective in labeling the smallest capillaries. Beyond its materialproperties, its main attraction for this application is that it can bemixed with fluorescent dyes and made extremely fluorescent. Thus,preferred embodiments utilize a fluorescent casting material.

The resin has been used with a multi-photon imaging system by performingwhole body perfusion castings of both mice and rabbits. The casting isfollowed by a perfusion and fixation protocol. For the mouse casting,access is gained through the apex of the left ventricle of the heartwith a 21-gauge cannula and secured in place with super glue. The rightatrium is punctured to allow outflow of the perfusate. All perfusionsolutions are warmed to 37° C. in a water bath prior to use. Perfusion(˜20 ml) physiological NaCl is performed until all blood is flushed fromthe animal. Following, the tissue is fixed by perfusion with 20 ml of 4%paraformaldehyde (PFA) in PBS, through the systemic circulation.Intravascular fixation is followed by the casting material.

Immediately after the perfusion and fixation the cast is mixed withhardener (0.8 ml) to the mixture of PU4ii resin (5 ml), solvent(2-Butanone (MEK)) (4 ml) and dye (7.5 g). The liquid is stirred brieflyand then injected into the animal through the same cannula the perfusionand fixation was performed. To remove small air bubbles in the resin,the final mixture is set under vacuum for 2-3 minutes using a standardvacuum chamber. At the time of injection the casting material has aconsistency of water.

Using the vibratome and tissue retrieval system images are taken througha 5 mm×5 mm×5 mm portion of tissue with 1 μm radial resolution and 2 μmaxial resolution in under 2 hours and recover the skeletal geometry ofthe vasculature tree with an automated thresholding algorithm.

Previous studies of infarct models have shown that the pro-angiogenicfactor bFGF administered via a sodium alginate polymeric patch induces astrong angiogenic response in treated rabbits versus untreated controls.A marked increase in vascular density near the region of the patch wasobserved by Day 2 and 8 (FIG. 12C). By Day 30 however, the vasculardensity (FIG. 12B) tended to regress to initial levels (FIG. 12C). Theseresults were obtained by histological examination of the heart tissueafter imaging and sectioning (FIG. 11). Due to the time consuming natureof the inspection procedure, four 10 μm thick sections were chosen at 2mm intervals from the apical end (FIG. 12A). These sections were IHCstained for endothelial cells and the vasculature density established bymanual counting.

There were several limitations of the analysis. Traditional histologicalexamination of IHC stained tissues is time consuming and became the ratelimiting step. IHC patterns were often difficult to interpret andconsistent results between different examiners were hard to obtain,making comparison between different studies at different timesproblematic. Additionally, with IHC staining all endothelial cells arelabeled, including both patent and non-patent vessels. Further, 2Dsections sacrifice all information about the 3D vascular network. Thesefactors lead to difficulties in using the recovered vasculature patternsfor models of vascular flow and drug uptake, which in turn severelyhindered the development of new delivery strategies.

This process can confirm the results for Day 2, 8, 30 shown in FIG. 13,where an initial increased vascular density was observed whichsubsequently decreased to initial levels by day 30.

Besides the vascular network, the system can image nuclei, tissuecollagen content (through SHG imaging), and tissue autofluorescence. Inaddition the hypoxia marker EF5 can be imaged on every tenth section (1mm) via IHC staining of the recovered slices. This provides a total ofat least 5 spectroscopically distinct components.

In-Vivo Myocardial Drug Delivery New Zealand White rabbits (3-3.5 kg)can be anesthetized with an intramuscular injection of 35 mg/kg ketamineand 5 mg/kg of xylazine. Rabbits are intubated with a 3.0 mmendotracheal tube for inhalant anesthetics and positive pressureventilation. The level of anesthesia is maintained with isofluoranebetween 1-3%. The skin over the chest is shaved and sterilely preparedwith Betadine and alcohol. In preparation for a thoracotomy, apre-emptive line block of Lidocaine will be administered along thesurgical incision. A left thoracotomy will be performed to gain accessto the heart. A clamp is used to keep the chest open throughout theprocedure. A small opening in the pericardium can be created while carewill be taken to minimize the damage to the pericardium. The leftanterior descending coronary artery is located and ligated with a sutureto create an ischemic heart model, with successful induction of ischemiadocumented by elevations of the ST segments on simultaneous continuousECG recording. bFGF bound heparin sepharose beads can be placed in thepericardial space. The pericardium is kept closed with suture to preventleakage of bFGF. The thoracotomy and skin incision is then be closed. Apositive end expiratory pressure and negative pressure chest tube willbe used during closure to prevent pneumothorax. An injection of 0.03mg/kg buprenorphine will be given subcutaneously every 8 hours for first72 hours for analgesia. Control animals receive heparin sepharose beadsembedded sodium alginate polymeric devices without growth factor.

The hearts are then dissected from the animal and perfusion casted withPU4ii through the main artery as is described herein. In addition thenuclei are perfusion labeled with 20 ml Hoechst 33342 (100 micro g/ml).The Hoechst labeling step is included in the perfusion procedure afterthe heart was flushed with PBS but before it is fixed and casted. It isnot necessary to label collagen as that will be detected by intrinsicsecond harmonic generation (SHG). In order to directly assess tissuehypoxia states we will also treat the animal with 2.0 mL of 10 mmol/LEF5 i.p three hours before sacrifice to enable later IHC stain for EF5.

IHC Staining: EF5 localizes to hypoxic tissue regions and has been usedextensively for labeling of hypoxia in both animal and tumors. Afteradministration to the animal, it is detected by monoclonal antibodies.The tissue sections are fixed in acetone for 10 minutes. Sections arethen washed in PBS and blocked with a protein-blocking reagent (ID Labs,Inc., London, Ontario, Canada) for 15 minutes to prevent nonspecificantibody binding. They are stained with a cyanine-5-conjugated mouseanti-EF5 (1/50) antibody for 1 hour in a humidified chamber (antibodydilutions indicated in parentheses). Finally, sections are washed in PBSfor two-photon fluorescent imaging. Cy5 fluorescence, representinghypoxic regions will be imaged with 665 to 695 nm emission filters.

Second Harmonic Generation (SHG) Imaging of Collagen: A key strength ofmultiphoton imaging is the ability to image elastin and collagen withina tissue without the need for exogenous labeling by exploiting the SHGsignal that is generated from the laser excitation. SHG is a scatteringprocess and therefore emits light at exactly half the excitationwavelength. A narrow band reflection filter extracts the SHG signalclearly from the remaining signals. Of particular note with regards tothis method, the system has the ability to image collagen remodeling inthe vascular tissue in response to angiogenesis, and collagen can beimaged in a mouse infarct in backscatter mode. The changes in the heartfunction in response to increased collagen deposition can haveclinically important ramifications. This is the first assay method toprovide information about this critical aspect of angiogenesis andischemia at the microscopic 3D level.

To perform this measurement, two treated and two control rabbits at eachtime point of day 2, 8 and 30 are used in the above procedure. Therabbit heart can be sectioned 8-10 mm from the apical end embedded in 7%agarose. Good results has been obtained with this concentration ofagarose for vibratoming of rabbit heart sections of 100 microns whichhave been cast with PU4ii. The tissue block can be mounted on the 3Dtissue cytometer and 3D images can be acquired according the procedureexplained herein.

To demonstrate the ability to overlay IHC stained images onto theoriginal data set, the system retrieves every tenth 100 μm thick section(total 6-8 sections) for IHC staining which has been transferred fromthe tissue with the tissue section retrieval module described herein.The system incorporates automated transfer of each section to an indexedeppendorf tubes. The position is logged with respect to the entire 3Dstack, and IHC stained. Afterwards each individual capture slice ismounted and reimaged on the MMM system and the resulting images overlaidonto the originals.

As stated above, 5 components are examined:

TABLE B2 Component Label Wavelength Significance Vasculature PU4iiCasting 600 Angiogenic response Collagen SHG/intrinsic 400Fibrosis/Infarct region Nuclei Intravital 480 Tissue state/necrosisHoescht 33342 EF5 Injection 650 Tissue Hypoxia followed by IHCAutofluorescence Intrinsic 430-650 Tissue ultrastructure

3D Microvascular Density:

To confirm the changes observed previously in vascular density atvarious time points, calculate the distance to the nearest blood vesselto each point with a custom algorithm (as illustrated in FIGS. 14A and14B). FIG. 14A shows the axial vessel orientation and FIG. 14B shows thelateral vessel orientation. A S/N ratio of 20-30 for the PU4ii cast makeit possible to threshold and segment the vasculature without resortingto computationally intensive semi-manual algorithms. From this, thedistance and geometry of tissue constituents such as cellular nuclei orcollagen can easily be mapped with relation to the vasculature. Thesteps in the method are: 1. Intensity illumination correction; 2. Medianfilter noise reduction; 3. Thresholding; 4. Morphological closing toremove objects smaller than a few pixels; 5. 3D distance transform.

The present application has shown data for staining of the nuclei inmouse heart with intravital labeling using Hoescht 33342. The nucleistained strongly and were straightforward to segment from the backgroundheart tissue even in the presence of vascular staining and tissueautofluorescence. Additionally, using multiphoton tissue cytometry toconduct 3D image cytometric measurement on large populations of cells upto 10⁵ provides quantitative measurement on these cell populationswithin a 3D matrix. The cellular density can be calculated by segmentingthe nuclei and then calculating the number of nuclei per unit volume asa function of position. The nuclei segmentation steps are 1. Automatedthreshold 2. Morphological opening/closing to remove spuriousconnections between nuclei 3. Morphological labeling to calculatenuclear volume and to assign an unique label to each nuclei candidate 4.Gating of nuclei targets that are outside the expected nuclear volume(500-3000 um³).

Collagen content, hypoxia state and tissue ultrastructure can beidentified spectrally and overlaid onto the segmented vascular images.

1. For the animals treated with bFGF, by calculating the averagedistance to the nearest blood vessel at each time point, demonstrate thevascular density trend displayed. One way ANOVA analysis of the meandistance can be used and a P<0.05 can be considered statisticallysignificant.

2. For the untreated controls, the method demonstrates no increase inthe vasculature density for Day 2 or Day 8 in comparison with Day 30.One way ANOVA analysis of the mean distance can be used and a P<0.05 canbe considered statistically significant.

3. The system provides the average distance from the hypoxia borderregion to the vasculature for the treated animals and untreatedcontrols.

4. To gauge tissue viability, the system calculates a correlationcoefficient of the degree of overlap of the nuclei density and hypoxicregions. A student t-distribution and a P<0.05 can be consideredsignificant.

An additional application is with regards to the production of brainatlases. Brain atlases are as essential for the research neuroscientist.Among the advantages of electronic atlases, compared with conventionalpaper atlases, are that an electronic atlas can contain images of allsections, allowing the brain to be “re-sectioned” in any desired plane,can offer multiple levels of resolution and a range of colors, canpresent structural features in 3D with variable transparency of surfaceand internal components and free rotation so as to optimize the user'sview of structural relationships, can provide labels at the whim of theuser in more than one language, is readily edited and updated, presentsflexible indexing and ready access to other pertinent databases.

The complexity of the mammalian brain makes it necessary to rely on mapsand atlases to analyze and interpret observations effectively. Modernneuroanatomic digital atlases are based on multiplemodality datasetsthat include histology, immunohistochemistry, magnetic resonanceimaging, positron emission tomography and three-dimensional (3D)reconstruction to describe structures, nuclei and connectivity. With theadvent of large-scale gene expression profiling using microarrays andhigh-throughput in situ hybridization in the present invention can buildan anatomical brain atlas based on the transcriptome alone or using itas a key supporting modality. The brain exhibits complex andcombinatorial gene expression patterns with variations depending on itshighly differentiated structure. Expression patterns of individual genesin the cerebral cortex and other brain structures have been shown tohighlight useful genetic markers for anatomic regions, boundaries,gradients and cell types. Profiling of larger brain structures usinglaser-capture microdissection and microarrays has suggested that geneexpression patterns established during embryogenesis are largelyretained in the adult and are important for regional specificity and forthe functional connective relationships between brain regions.Combinatorial gene expression patterns have been found to define adiversity of neural progenitor domains that yield particular functionalcomponents in the mature brain. It follows that combinatorial geneexpression characteristics should be reflected directly or indirectly inthe neuroanatomic organization in the adult. Through the synthesis of insilico expression patterns across many genes in a spatially aligneddataset, an enhanced understanding of the relationships between genesexpressed and structural and functional neuroanatomy may emerge.

Given the usefulness of brain atlases, there is a strong need for robustmethods to quickly produce atlases of mouse brain for gene expressionstudies in automated manner. Unfortunately, the current method ofemploying serial section analysis is time consuming and labor intensiveand often result in low quality datasets. However, using the whole mounttissue scanner method described herein, the present invention canquickly generate mouse brain atlases in an automated fashion.

Referring to the flowcharts in FIGS. 6-8, details of the steps involvedin the entire process from mounting the mouse brain sample, imaging andsectioning, capturing of slices, processing of slices with more detailedanalysis, re-imaging of the slices, and registration of slices back tothe whole mount dataset.

FIG. 15 is a diagram showing the general geometry of the alternationbetween optical imaging and a depth of, say, 50 μm, into the tissue, andmechanical sectioning, of say, 100 μm. Portions of a resulting datasetis shown in FIGS. 16A-16D.

The brain can be fixed with a perfusion fixation in a paraformaldehydeand then embedded into agarose block to improve mechanical stabilityduring the sectioning process. The sample is mounted in a water bath tokeep the tissue hydrated and to lubricate the cutting process by thevibratome. See FIG. 9 and the description thereof.

Mouse brain atlases with pixel samplings from 0.1 μm to 2.0 μm arepossible to obtain with the optomechanics current implementation of thistechnique. Individual images with pixel dimensions of 416 to 2080 can beproduced and physical dimensions from 200 μm to 2500 μm. See FIGS.17A-17D. These individual images are filed and registered together toform a larger montage of a mouse brain sections. The sections can thenbe aligned with respect to one another to produce a mouse brain atlas.See FIG. 18.

Mechanical sections of 40 μm are greater is feasible. It is important tonote that tissue sections on the size of 40 μm are near ideal for IHCstudies. Optical section within the mechanical section on with z-spacingof 2 μm is also possible. Multichannel acquisition is also possible. Togenerate a brain atlas of 1.4 μm pixel sampling, at 100 micron coronalsection interval, a time of 4 hours is required.

FIGS. 15 and 16A-16D show an example of the sort of datasets that can begenerated with preferred embodiment of the invention.

Referring to FIG. 10 showing individual brain sections which have beencaptured and transferred to their own chamber in a well plate with anarray 820 of single section chambers.

The next step after sectioning is to process each individual slice. Asan example, we will use the IHC staining of each brain section as theprocessing example Immunocytochemistry comprises a number of methods,where antibodies are employed to localize antigens in tissues or cellsfor microscopic examination. There are several strategies to visualizethe antibody. For transmitted light microscopy, color developmentsubstrates for enzymes are often used. The antibody can be directlylabeled with the enzyme. However, such a covalent link between anantibody and an enzyme might result in a loss of both enzyme andantibody activity. For these reasons several multistep stainingprocedures have been developed, where intermediate link antibodies areused. In this protocol, we use the Vectastain ABC-kit. In the laststaining step, the reaction is visualized with a 3-3′ diaminobenzidinetetrahydrochloride (DAB).

There are several methods to IHC stain each brain section. The procedureinvolves free floating IHC staining of tissue sections as an example. Anexample free floating staining protocol can be found in Pete et al. 2002part of which is below:

The free-floating sections were washed in phosphate-buffered saline(PBS) containing 0.3% Triton-X, and then 1-in-5 series of sections wasxposed for 30 min to PBS-Triton solution containing 3% normal rabbitserum, to block nonspecific binding sites. After a further wash, thetissue was placed overnight at room temperature in a primary polyclonalantibody solution (1:10,000 dilution of rabbit anti-Fos in PBS;Oncogene). The sections were rinsed, incubated with biotinylated goatanti-rabbit secondary antiserum and further processed using the standardbiotin avidin-peroxidase kit (Vector, ABC-elite kit). The immunoreactionwas visualized by incubating the sections with 0.02%3,3_-diaminobenzidine containing 0.01% hydrogen peroxide for 6 min Apurple-black reaction product was obtained by adding nickel chloride tothe peroxidase reaction (40_1 of 8% NiCl2 solution per 100 ml of DABsolution), as previously described (Haxhiu et al., 1996; Belegu et al.,1999, incorporated herein by reference). Subsequently, the sections werewashed in PBS (2×), mounted on poly-L-lysine-coated slides, and preparedfor in situ hybridization.

Once the brain tissue has been IHC stained, it can be reimaged by avariety of methods including wide field, multiphoton and confocalmicroscopy. Once reimaged, it can be registered and morphed onto theoriginal dataset by a variety of registration and morphing algorithms.This process can be done manually or automatically by a computer. Inthis manner a 3D IHC stained dataset of mouse brain atlas can beproduced.

While this invention has been particularly shown and described withreference to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details can bemade without departing from the scope of the invention encompassed bythe appended claims.

The invention claimed is:
 1. A method of imaging tissue comprising:illuminating a first region of the tissue with light; detecting lightfrom the tissue in response to the illuminating light to form at least afirst image of the first region of the tissue wherein detected lightincludes light received from a depth in the tissue below a firstsectioning depth to provide first registration data; sectioning aportion of the tissue at the first sectioning depth to expose a secondregion of the tissue with a sectioning tool; illuminating the secondregion of the tissue; detecting light from the second region of thetissue to form at least a second image of the second region of thetissue; sectioning at least a portion of the second region of the tissuewith the sectioning tool at a second sectioning depth wherein detectedlight includes light received from a depth in the tissue below thesecond sectioning depth to provide second registration data; processingthe images of the first region of the tissue and the second region ofthe tissue including at least a first optical tomography image of thefirst region of the tissue and a second optical tomography image of thesecond region of the tissue; and indexing the images using the firstregistration data and the second registration data to form a threedimensional image formed with at least the first optical tomographyimage of the first region of the tissue and the second opticaltomography image of the second region of the tissue.
 2. The method ofclaim 1 further comprising staining a first section of the tissue and asecond section of the tissue.
 3. The method of claim 2 wherein thestaining step uses immunohistochemistry (IHC) staining.
 4. The method ofclaim 1 further comprising processing the tissue with a processcomprising one or more of staining analysis, mass spectrometry, microdissection, and imaging mass spectrometry.
 5. The method of claim 1wherein the step of detecting light to form the image of the firstregion of the tissue further comprises imaging the first region of thetissue with a first imaging process.
 6. The method of claim 5 furthercomprising imaging a first processed section of the tissue wherein saidimaging of the first region of the tissue further comprises imaging witha second imaging process different from the first imaging process. 7.The method of claim 6 further comprising imaging the first region of thetissue with a two photon microscopy system.
 8. The method of claim 6further comprising imaging the first sectioned portion of the tissuewith a confocal microscopy system.
 9. The method of claim 6 wherein theilluminating steps comprise using a first excitation wavelength and asecond excitation wavelength.
 10. The method of claim 6 furthercomprising imaging a plurality of processed sections by detecting adifferent emission wavelength of light from each processed section. 11.The method of claim 1 wherein the tissue sample includes a vasculartissue such that vascular features of the image of the first region ofthe tissue are correlated with the vascular features of a processedtissue image.
 12. The method of claim 1 wherein nuclei in the firstimage of the first region of tissue are correlated with the nuclei in aprocessed image of the first region of tissue.
 13. The method of claim 1wherein neurons are used to correlate a pre-sectioned region of thetissue with a post-sectioned region of the tissue.
 14. The method ofclaim 1 further comprising using an anatomical structure in an image ofa region of a brain to correlate different images of the region of thebrain.
 15. The method of claim 1 further comprising registering an imageof a processed section of the tissue to a pre-sectioned image of thetissue.
 16. The method of claim 1 further comprising imaging tissueincluding imaging an entire animal organ.
 17. The method of claim 1further comprising storing images for a brain atlas.
 18. The method ofclaim 1 wherein the step of sectioning the tissue further comprisestranslating a sectioning tool to remove a section from the sample. 19.The method of claim 1 further comprising imaging processed sections byselecting an imaging modality comprising one or more of opticalcoherence tomography imaging (OCT), optical projection tomographyimaging, confocal imaging, second harmonic generation imaging (SHG),wide field imaging and time resolved fluorescence imaging.
 20. Themethod of claim 6 further comprising imaging a vascular cast.
 21. Amethod of imaging brain tissue comprising: illuminating a first regionof the brain tissue with light; detecting light from the brain tissue inresponse to the illuminating light to form at least a first image of thefirst region of the brain tissue; sectioning a portion of the braintissue to expose a second region of the brain tissue with a sectioningtool; positioning the exposed second region of the brain tissue relativeto a scanning lens using a translation stage connected to an automatedcontrol system; illuminating the second region of the brain tissue;detecting light from the second region of the tissue to form at least asecond image of the second region of the tissue; sectioning at least aportion of the second region of the brain tissue with the sectioningtool; and processing the images of the first region of the brain tissueand the second region of the brain tissue including at least a firstoptical tomography image of the first region of the brain tissue and asecond optical tomography image of the second region of the brain tissuewith a data processor.
 22. The method of claim 21 further comprisingindexing the first optical tomography image to form a three dimensionaloptical tomography image.
 23. The method of claim 21 wherein thesectioning tool comprises a microtome.
 24. The method of claim 21wherein the sectioning tool comprises a vibratome.
 25. The method ofclaim 21 further comprising processing image data with a data processorthat processes image data of a first sectioned tissue surface tocorrelate features of the first sectioned tissue surface with featuresof a second tissue surface that has been separated from.
 26. The methodof claim 21 further comprising stacking and aligning the images to forma three dimensional (3D) image.
 27. The method of claim 21 furthercomprising registering images using vascular features within the images.